Dissociated cell cultures of fetal mouse spinal cord (SC) and dorsal root ganglion (DRG) neurons have been used in conjunction with a 3-compartment culture system to study the process of activity-dependent synapse augmentation and elimination. With this system, two independent populations of DRG neurons converge on and synapse with a common population of SC neurons. Chronic (3-4 days) electrical stimulation of one set of DRG axons results in this population establishing stronger synaptic connections with the target SC neurons relative to the convergent non-stimulated DRG axons. The NMDA receptor antagonist, APV, blocks this plasticity, but in solutions with raised calcium ion concentration (3 mM vs. 1.8 mM) plus AVP, stimulation again produces stronger synaptic connections from the stimulated axons as compared to those from the non-stimulated convergent axons. We infer a critical role for an increased intracellular calcium in the process of activity-dependent synapse competition.